General base catalysis by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus: the role of Glu4 and Asp55.
General base catalysis by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus: the role of Glu4 and Asp55.
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来自蜡样芽孢杆菌的磷脂酰胆碱偏好性磷脂酶 C 的一般碱催化：Glu4 和 Asp55 的作用。
DOI：
10.1021/bi972948k
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Hergenrother,PJ
中科院分区：
文献类型：
--
作者：
Martin,SF;Hergenrother,PJ
为了评估蜡样芽孢杆菌(PLCBc)的磷脂酰胆碱偏好性磷脂酶C (PLCBc)的活性位点残基Glu4和Asp55在结合和催化中可能发挥的作用，通过plc基因的定点诱变制备了选定的突变体。 The mutants were then expressed inEscherichiacoliand purified as fusion proteins with the maltose binding protein (MBP).动力学分析表明，Glu4 突变对催化活性影响不大，而 Asp55 突变导致蛋白质的 forkcat/KM 值比野生型酶低 104−106 倍。 E4L 突变体催化活性的适度下降和 pH 依赖性特征强烈表明，4 位谷氨酸不是 PLCBc 催化反应中的通用碱基。相反，结果支持这样的假设：Glu4 主要参与底物结合，可能是通过静电稳定磷脂酰胆碱底物的胆碱部分的正电荷。对 PLCB 及其各种配合物的 X 射线晶体学数据的检查表明，Asp55 的羧酸侧链的位置使得它可以激活水以对底物进行亲核攻击或充当 Zn1 的配体。然而，Asp55 侧链作为重要的 Zn1 配体的参与与 D55L 突变体获得的原子吸收和热稳定性数据不一致，这些数据与野生型酶的原子吸收和热稳定性数据几乎相同。 PLCB 的 D55E、D55N 和 D55L 突变体的测量 kcat/KM 大幅降低，表明 Asp55 在催化中发挥着关键作用，并且可能作为 PLCBc 水解磷脂酰胆碱的通用基础。
To assess what roles the active site residues Glu4 and Asp55 of the phosphatidylcholine-preferring phospholipase C ofBacilluscereus(PLCBc) might play in binding and catalysis, selected mutants were prepared through site-directed mutagenesis of theplcgene. The mutants were then expressed inEscherichiacoliand purified as fusion proteins with the maltose binding protein (MBP). Kinetic analysis showed that mutations at Glu4 had only modest effects on the catalytic activity, whereas those at Asp55 led to proteins whose values forkcat/KMwere 104−106times less than that of the wild-type enzyme. The modest decrease in catalytic activity and the pH-dependent profile of the E4L mutant strongly suggest that glutamic acid at position 4 is not the general base in the PLCBc-catalyzed reaction. Rather, the results support the hypothesis that Glu4 is primarily involved in substrate binding, perhaps by electrostatic stabilization of the positive charge of the choline moiety of the phosphatidylcholine substrate. Examination of X-ray crystallographic data of PLCBcand its various complexes reveals that the carboxylate side chain of Asp55 is positioned such that it could activate a water for nucleophilic attack on the substrate or serve as a ligand for Zn1. However, the involvement of the side chain of Asp55 as an important Zn1 ligand is not consistent with the atomic absorption and thermostability data obtained for the D55L mutant, which are virtually identical with that of the wild-type enzyme. The large reduction in the measuredkcat/KMof the D55E, D55N, and D55L mutants of PLCBcindicates that Asp55 plays a critical role in catalysis and likely serves as the general base in the hydrolysis of phosphatidylcholine by PLCBc.